Hepatitis B virus (HBV) is a hepatotropic DNA virus that replicates by reverse transcription. It chronically infects >350 million people worldwide and kills up to 1.2 million patients annually. Therapy employs nucleos(t)ide analogs that suppress viral DNA synthesis very well, but they cure only a few percent of patients even after years of treatment. Importantly, the ability of the nucleos(t)ide analogs to cure some patients indicates that they can push the virus to the brink of elimination. This presents a huge opportunity to cure many patients by suppressing HBV further. All HBV DNAs are produced by reverse transcription in the cytoplasm through the action of 2 viral enzymatic activities, the DNA polymerase that synthesizes the DNA and the ribonuclease H (RNAse H) that destroys the viral RNA after it has been copied into DNA. Newly synthesized genomes can either be enveloped and secreted from the cell as virions, or they can be transported into the nucleus to replenish the episomal form of the viral genome (the cccDNA). The cccDNA is key to maintenance of HBV infection because it is the template for HBV transcription, and curing HBV means eliminating the cccDNA. The indefinite persistence of the cccDNA in patients whose viral titres in serum have been suppressed below the limit of clinical detection by the nucleos(t)ide analogs is the result of residual viral replication. This leads to replenishment of the hepatic nuclear cccDNA pool by intracellular genomic transport and low-level infection of new cells. Therefore, novel drugs that act on targets other than the DNA polymerase active site are urgently needed. These drugs would be used together with the existing nucleos(t)ide analogs to suppress HBV replication below the level needed to maintain the cccDNA. The HBV RNAse H activity has not been a focus of drug development despite being an attractive target because solubility and stability problems prevented production of active RNAse H suitable for drug screening. We can now produce active recombinant HBV RNAse H. Here, we will work closely with the professional drug discovery team at Saint Louis University to develop a high-throughput screen (HTS) for RNAse H inhibitors in preparation for anti-HBV RNAse H drug development. This HTS will be coupled with our existing biochemical and cell-based RNAse H assays to provide an integrated biochemistry-to-cell culture pipeline for development of novel anti-HBV RNAse H drugs. Aim 1. HTS assay development and performance validation. Our RNAse H assay will be adapted to a fluorescent format and miniaturized based on similar assays previously used to identify anti-HIV RNAse H compounds. The performance characteristics of the assay under HTS conditions will be established. Aim 2. Proof-of-principle HTS for anti-HBV RNAse H compounds. 6588 compounds will be screened to demonstrate robustness of the assay and to guide library selection for future drug screening.